The present invention relates generally to light detectors and more particularly to a system for measuring the decay time constant of light emitted from a molecule after it is excited by a pulse of light or electric current.
Luminometry is a highly sensitive detection technique to detect the presence or concentration of a specific material, such as bio-medical molecules. However, the luminescent molecules can be very sensitive to environment variations, and most substances emit light that contributes to an unpredictable background luminance. Time-resolved spectroscopy is attractive because a molecule is identified by its specific decay constant that is not sensitive to the environment condition. With background luminance quickly reduced, the delayed fluorescence technology is a contrast enhancing mechanism.
The conventional time-resolved spectroscopic detection system requires a short pulsed light, or electric source and a xe2x80x9cgatedxe2x80x9d detector. In a typical time resolved fluorescence microscopy, a gated detector is synchronized, with controllable delay time, to the excitation of the specimen by a laser, a flush lamp, or a pulsed LED (light emitting diode). An intensified CCD (charge-coupled device) camera can be easily gated for time resolved fluorescence study. Use of the intensified CCD camera, however, causes a deteriorating signal to noise ratio, because the thermal noise from the photocathode as well as electron multiplication noise from the microchannel plate reduces the signal to noise ratio. In addition, image intensifiers cause a reduction in the intrascene dynamic range.
Other gating technologies have their drawbacks. A gated photomultiple tube may also be used as a gated detector. However, the signal is read point by point with this method. A rotating wheel with holes is another gating technology used in time-resolved spectroscopy. Application of this slow gating technology is limited to molecules with a very long decay lifetime. With conventional xe2x80x9cgatingxe2x80x9d technology, such as intensified CCD, the signal is observed only at the xe2x80x9cgated time intervalxe2x80x9d for each exciting pulse. Most of the fluorescent light that falls outside the time gate is wasted.
Other time-resolved fluorescence detection methods include modulating the exciting light and deriving the decay time constant from the phase shift of the modulated signal relative to the exciting light, or deriving the decay time constant by studying the polarization measurement. Each of these methods has drawbacks that are well known by practitioners in the art.
The main difficulty in time-resolved fluorescence spectroscopy is therefore developing a suitable light measurement technology for luminance decay detection over time. Accordingly, the need remains for overcoming problems inherent with time resolved fluorescence spectroscopy and to develop a suitable time-resolved light detection system.
The present invention relates to a technology that is based on the conversion of a slit in space to a gate in time to perform time resolved spectroscopy.
In an embodiment of this invention, the measurement system of the present invention utilizes a CCD (charge coupled device) camera to measure the intensity of the light emitted from a sample. The CCD camera has rows and column of pixels. Charge stored in the pixels can be transferred in parallel rows. In a preferred implementation of the invention, the CCD pixels are covered with a mask that has slits. When the sample under study is illuminated with a short light pulse and fluoresces, an image of the sample forms on the mask. With the mask blocking most of the area, only pixels underneath the slit are exposed to the fluorescent light. When charges in the pixels are transferred in parallel in a certain direction at a constant rate, the whole decay curve of the fluorescence can be detected with a single exciting light pulse.
The proposed technology will therefore have the advantage in efficiency over existing technologies. More importantly, with the whole decay curve recorded with each single pulse, xe2x80x9cnoisesxe2x80x9d caused by fluctuation in the power of an individual exciting light pulse, which happens in all of the gated detection system, is eliminated.